Abstract
Reverse genetics in Xenopus has been limited to knockdown strategies using antisense morpholino oligonucleotides (MOs). Recently, engineered zinc-finger nucleases have been used to induce targeted mutations resulting in null alleles. Zinc-finger nuclease (ZFN) technology has been adapted to induce null mutations in many systems previously refractory to targeted gene inactivation. Here we provide a general protocol for inducing targeted mutations in Xenopus tropicalis using ZFNs, a method to detect resulting mutations, and the steps to generate homozygous mutant embryos.
MeSH terms
-
Animals
-
Animals, Inbred Strains
-
Chorionic Gonadotropin / administration & dosage
-
Cloning, Molecular
-
DNA Mutational Analysis
-
Deoxyribonucleases / chemistry
-
Deoxyribonucleases / genetics*
-
Embryo, Nonmammalian / physiology
-
Female
-
Fertilization
-
Germ-Line Mutation
-
Homozygote
-
INDEL Mutation
-
Male
-
Mutagenesis
-
Protein Engineering
-
Recombinant Fusion Proteins / chemistry
-
Recombinant Fusion Proteins / genetics
-
Reproductive Control Agents / administration & dosage
-
Reverse Genetics*
-
Xenopus / genetics*
-
Zinc Fingers
Substances
-
Chorionic Gonadotropin
-
Recombinant Fusion Proteins
-
Reproductive Control Agents
-
Deoxyribonucleases